Genotypic Diversity Is Associated with Clinical Outcome and Phenotype in Cryptococcal Meningitis across Southern Africa.

Mathew A Beale,Simon J O’Hanlon,Robin C May,Emma J Robertson,Wilber Sabiiti,Angela Loyse,Matthew C Fisher,Tihana Bicanic,Graeme Meintjes,Joseph N Jarvis,Thomas S Harrison,Karen M Fuentes-Cabrejo

doi:10.1371/journal.pntd.0003847

Abstract

Cryptococcal meningitis is a major cause of mortality throughout the developing world, yet little is known about the genetic markers underlying Cryptococcal virulence and patient outcome. We studied a cohort of 230 Cryptococcus neoformans (Cn) isolates from HIV-positive South African clinical trial patients with detailed clinical follow-up using multi-locus sequence typing and in vitro phenotypic virulence assays, correlating these data with clinical and fungal markers of disease in the patient. South African Cn displayed high levels of genetic diversity and locus variability compared to globally distributed types, and we identified 50 sequence types grouped within the main molecular types VNI, VNII and VNB, with 72% of isolates typed into one of seven 'high frequency' sequence types. Spatial analysis of patients’ cryptococcal genotype was not shown to be clustered geographically, which might argue against recent local acquisition and in favour of reactivation of latent infection. Through comparison of MLST genotyping data with clinical parameters, we found a relationship between genetic lineage and clinical outcome, with patients infected with the VNB lineage having significantly worse survival (n=8, HR 3.35, CI 1.51-7.20, p=0.003), and this was maintained even after adjustment for known prognostic indicators and treatment regimen. Comparison of fungal genotype with in vitro phenotype (phagocytosis, laccase activity and CSF survival) performed on a subset of 89 isolates revealed evidence of lineage-associated virulence phenotype, with the VNII lineage displaying increased laccase activity (p=0.001) and ex vivo CSF survival (p=0.0001). These findings show that Cryptococcus neoformans is a phenotypically heterogeneous pathogen, and that lineage plays an important role in cryptococcal virulence during human infection. Furthermore, a detailed understanding of the genetic diversity in Southern Africa will support further investigation into how genetic diversity is structured across African environments, allowing assessment of the risks different ecotypes pose to infection.

Highlights

Cryptococcal meningitis (CM), caused by the fungus Cryptococcus neoformans (Cn) is one of the major causes of AIDS-related mortality, in sub-Saharan Africa, where it was estimated to account for as many as 500,000 deaths annually prior to wide scale availability of antiretroviral therapy (ART) [1]
Cryptococcus neoformans (Cn) is a yeast that commonly causes meningitis in HIV infected individuals in Africa, where it may account for up to 500,000 deaths every year. In this highly translational and multidisciplinary study, we used genetic analysis techniques to show that Cryptococcus found in Southern Africa represents a hotspot of genetic diversity
We combined this data with the results of microbiological techniques that assess the natural virulence traits that the yeast uses to survive and infect humans to further show that genetic diversity is associated with differences in cryptococcal phenotype

Summary

Introduction

Cryptococcal meningitis (CM), caused by the fungus Cryptococcus neoformans (Cn) is one of the major causes of AIDS-related mortality, in sub-Saharan Africa, where it was estimated to account for as many as 500,000 deaths annually prior to wide scale availability of antiretroviral therapy (ART) [1]. A number of clinical adverse prognostic markers in HIV-associated CM have been identified, including high fungal burden at CM diagnosis, poor rate of cryptococcal clearance from patient cerebrospinal fluid (CSF) during antifungal treatment, and altered mental status at presentation [5,6]. These clinical parameters are associated with poor outcome, the mechanisms underpinning this variation have not yet been fully explored. We recently demonstrated an association between the in vitro phagocytosis of clinical cryptococcal isolates by J774 murine macrophage-like cells and poor patient survival in a cohort of 65 patients with HIV-associated CM [8] These phenotypic differences are likely the result of genetic variation between isolates. Previous studies in other pathogenic microorganisms and fungi have shown evidence of genetic lineage associated with phenotype and clinical outcome [10,11,12], and a recent study of 140 Cn isolates from Uganda provided the first evidence of this in Cryptococcus [13]

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Results

Discussion

Conclusion